Biomarkers associated with nephropathy

ABSTRACT

Use of urine biomarkers for diagnosing nephropathy, monitoring nephropathy progress, and assessing efficacy of a nephropathy treatment. These urine biomarkers include leukocyte-associated Ig-like receptor-2, alpha-1 acid glycoprotein, their fragments, and combinations thereof.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/969,731, filed on Dec. 16, 2010, which is a continuation of U.S.application Ser. No. 12/694,639, filed on Jan. 27, 2010, which claimspriority to U.S. Provisional Application No. 61/147,785, filed on Jan.28, 2009. The contents of all prior applications are hereby incorporatedby reference in their entireties.

BACKGROUND OF THE INVENTION

Nephropathy, commonly known as kidney damage, is caused by, amongothers, diabetes, high blood pressure, drug toxicity, and inflammation.

Typically, nephropathy is diagnosed by determining the level ofproteinuria (e.g., the level of urine albumin), or by examining theglomerular filtration rate (GFR), an indicator of renal function. Bothapproaches are not suitable for detecting early stage nephropathy, whichtypically displays no symptoms. While nephropathy can also be detectedby renal biopsy, this invasive procedure is not an ideal diagnosticapproach.

It is of great importance to develop a method for detecting early stagenephropathy. The key to achieving this goal is to identify reliablebiomarkers associated with incipient nephropathy.

SUMMARY OF THE INVENTION

The present invention is based on unexpected discoveries that the urinelevels of leukocyte-associated Ig-like receptor-2, alpha-1 acidglycoprotein, and fragments of these two proteins are significantlyhigher in a nephropathy patient than in a nephropathy-free patient.These protein molecules are therefore reliable biomarkers for diagnosisof early stage nephropathy.

Accordingly, one aspect of this invention features a nephropathydiagnostic method. This method includes at least the following steps:(a) obtaining a urine sample from a subject suspected of havingnephropathy, (b) determining in the urine sample a level of a biomarker,and (c) assessing whether the subject has nephropathy based on the levelof the biomarker. The biomarker used in the just-described method is oneof the following: (i) leukocyte-associated Ig-like receptor-2 or afragment thereof having at least ten amino acid residues, such asDFLELLVKGTVPGTEASGFDAP (SEQ ID NO:1), (ii) a fragment of alpha-1 acidglycoprotein having at least ten amino acid residues, such asGQEHFAHLLILRDTKTYMLAFDVNDEKNWGLS (SEQ ID NO:2), (iii) a combination of(i) and (ii), or (iv) a combination of (i) and alpha-1 acidglycoprotein.

An increase in the level of one of the four biomarkers, as compared tothat in a nephropathy-free subject, indicates that the subject hasnephropathy. In one example, the biomarker level is determined by a massspectrometry assay (e.g., MALDI-MS, LC-MS, and LC-MS/MS). In anotherexample, it is determined by an immune assay (e.g., ELISA, Western blot,RIA, FIA and LIA).

The above-described nephropathy diagnostic method is applicable to bothhumans and laboratory animals, e.g., those free of proteinuria. The term“a laboratory animal” used herein refers to a vertebrate animal commonlyused in animal testing, e.g., mouse, rat, rabbit, cat, dog, pig, andnon-human primate.

Another aspect of this invention features a method for monitoringnephropathy progress in a subject. This method includes (a) obtaining afirst urine sample from a subject suffering from nephropathy (e.g., ahuman or a laboratory animal), (b) determining in the first urine samplea level of one of the four biomarkers listed above, (c) obtaining asecond urine sample from the subject 2 weeks to 12 months after thefirst urine sample is obtained, (d) determining in the second urinesample a level of the biomarker, and (e) assessing nephropathy progressin the subject. An increase in the level of the biomarker in the secondurine sample, as compared to that in the first urine sample, indicatesthat nephropathy is exacerbated in the subject. When the subject is ahuman in early stage nephropathy, the second urine sample is obtained 6to 12 months after the first urine sample is obtained. For a humansubject in late stage nephropathy, the second urine sample can beobtained 3 to 6 months later than the first urine sample. When thismethod is applied to a laboratory animal, the second urine sample can beobtained 2 to 24 weeks after the first urine sample is obtained.

In still another aspect, the present invention provides a method formonitoring efficacy of a nephropathy treatment in a nephropathy patient,including (a) determining a level of one of the biomarkers listed abovein a urine sample from the nephropathy patient before the treatment, (b)determining a level of the biomarker in a urine sample from the patientafter the treatment, and (c) assessing efficacy of the treatment basedon a change in the level of the biomarker after the treatment. Thetreatment is found to be effective when the post-treatment biomarkerlevel remains the same or decreases as compared with the pre-treatmentbiomarker level.

This invention also provides a method of assessing renal toxicity of anagent, including (a) obtaining a plurality of urine samples from asubject treated with an agent at various time points during treatment,(b) determining in each of the urine samples a level of one of theabove-described biomarkers, and (c) assessing renal toxicity of theagent based on a change in the level of the biomarker during thetreatment. An increase in the biomarker level in the course of thetreatment indicates that the agent is renal toxic. The agent can be acompound (e.g., a drug or a drug candidate), an herb product, and a foodproduct

This invention further provides a kit useful in any of the methodsdescribed above. This kit contains a first antibody specifically bindingto leukocyte-associated Ig-like receptor-2 and a second antibodyspecifically binding to alpha-1 acid glycoprotein. Both antibodies canbe whole immunoglobulin molecules. In one example, this kit containsonly antibodies specific to antigens to be detected (e.g., biomarkersassociated with nephropathy) for practicing one of the methods disclosedherein. Namely, it consists essentially of such antibodies.

Also within the scope of this invention is an isolated antibodyspecifically binding to DFLELLVKGTVPGTEASGFDAP (SEQ ID NO:1), orGQEHFAHLLILRDTKTYMLAFDVNDEKNWGLS (SEQ ID NO:2). The term “isolatedantibody” used herein refers to an antibody substantially free fromnaturally associated molecules. More specifically, a preparationcontaining the antibody is deemed as “an isolated antibody” when thenaturally associated molecules in the preparation constitute at most 20%by dry weight. Purity can be measured by any appropriate method, e.g.,column chromatography, polyacrylamide gel electrophoresis, and HPLC.

Any of the antibodies described above can be used in manufacturing a kituseful in practicing any of the methods of this invention.

The details of one or more embodiments of the invention are set forth inthe description below. Other features or advantages of the presentinvention will be apparent from the following detailed description ofseveral examples, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are first described.

FIG. 1 is a diagram showing boxplots for combined levels of a fragmentof leukocyte-associated Ig-like receptor-2 and a fragment of alpha-1acid glycoprotein in healthy controls and patients having various typesof nephropathy. DN, IgAN, CHN, and MGN refer to diabetic nephropathy,IgA nephropathy, Chinese herb nephropathy, and membranousglomerulonephritis nephropathy. The upper and lower limits of the boxesmark the 25% and 75% values with the medians as the lines across theboxes. The upper whisker marks the largest value below the upper fence,which is the 75% value plus 1.5 interquartile range and the lowerwhisker marks the smallest value above the lower fence, which is the 25%value minus 1.5 interquartile range.

FIG. 2 is a diagram showing boxplots for combined levels of a fragmentof leukocyte-associated Ig-like receptor-2 and a fragment of alpha-1acid glycoprotein in healthy controls and patients having CHN. The upperand lower limits of the boxes mark the 25% and 75% values with themedians as the lines across the boxes. The upper whisker marks thelargest value below the upper fence, which is the 75% value plus 1.5interquartile range and the lower whisker marks the smallest value abovethe lower fence, which is the 25% value minus 1.5 interquartile range.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention relates to a method for diagnosingnephropathy based on the level of a urine biomarker, which can beleukocyte-associated Ig-like receptor-2 (GenBank accession numberCAQ08962; 10 Jan. 2010), alpha-1 acid glycoprotein (GenBank accessionnumber EAW87416; 10 Jan. 2010), a fragment of either protein, or acombination thereof. The fragment of either protein has a minimum lengthof ten amino acids and preferably, a maximum length of 120 to 200 aminoacids. For example, fragments of leukocyte-associated Ig-like receptor-2and alpha-1 acid glycoprotein can contain up to 125 and 191 amino acidresidues, respectively. In one example, the fragment ofleukocyte-associated Ig-like receptor-2 is DFLELLVKGTVPGTEASGFDAP (SEQID NO:1) and a fragment of alpha-1 acid glycoprotein isGQEHFAHLLILRDTKTYMLAFDVNDEKNWGLS (SEQ ID NO:2).

Each of the urine biomarkers mentioned above can be used to diagnose anytype of nephropathy, including those related to diabetes (i.e., diabeticnephropathy), glomerulonephritis resulting from deposition of IgA inkidney tissues (i.e., IgA nephropathy), inflammation (e.g., membranousglomerulonephritis), Chinese herb-induced renal fibrosis (i.e., Chineseherbal nephropathy), chronic tubulointerstitial damage, which lead torenal failure (i.e., chronic interstitial nephritis), and focalsegmental glomerulosclerosis.

To practice the diagnostic method of this invention, a urine sample isobtained from a subject suspected of having nephropathy and the level ofany of the biomarkers mentioned above is then determined by conventionalmethods, e.g., ELISA and Western blot. When the biomarker is a peptideor a combination of peptides, its level can be determined by a massspectrometry assay. The level of the urine biomarker can then becompared with a reference point representing the level of the same urinebiomarker in a nephropathy-free subject. The reference point can bedetermined via routine practice based on the representative level of aurine biomarker in a group of nephropathy patients versus that in agroup of nephropathy-free subjects. For example, it can be the middlepoint between the mean levels of these two groups. When the level of theurine biomarker in the subject is greater than the reference point, itindicates that the subject has nephropathy.

When necessary, patients having minimal change nephropathy (MCN) orminimal change disease (MCD) can be used as control groups fordetermining the reference point mentioned above. Generally, MCN and MCDpatients exhibit obvious proteinuria but normal renal functions.

When a fragment of leukocyte-associated Ig-like receptor-2 or a fragmentof alpha-1 acid glycoprotein is used, the diagnostic method of thisinvention can be applied to detect incipient nephropathy when presenceof proteins (e.g., albumin) in urine is not detectable, i.e., free ofproteinuria.

In another aspect, this invention relates to a method of monitoringnephropathy progress in a subject based on any of the urine biomarkersdescribed above. To practice this method, two urine samples from asubject can be obtained within a suitable time span (e.g., 2 weeks to 12months) and examined to determine the levels of one of the urinebiomarkers described above. If the urine biomarker level in thelater-obtained urine sample is greater than that in the earlier-obtainedurine sample, it indicates that nephropathy progresses in the subject.

The monitoring method can be applied to a human subject suffering fromor at risk for nephropathy. When the human subject is at risk for or inearly stage nephropathy, the level of the urine biomarker can bedetermined once every 6 to 12 months to monitor nephropathy progress.When the human subject is already in late stage of nephropathy, it ispreferred that the urine biomarker level be determined once every 3 to 6months. While carrying kidney damage, patients in early stagenephropathy are generally asymptomatic and display normal kidneyfunctions. These patients are at risk for nephropathy progress. Laterstage nephropathy is characterized by a progressive decline in GFR(e.g., <15 mL/minute/1.73 m²).

The monitoring method described above is also applicable a laboratoryanimal, following routine procedures, to study nephropathy. Preferably,the laboratory animal is examined once every 2 to 24 weeks to determinethe level of one of the urine biomarkers mentioned above. An increase inthe biomarker level over time indicates that the disease progresses inthe animal.

In yet another aspect, the present invention provides a method forassessing efficacy of a nephropathy treatment in a subject in need(i.e., a human nephropathy patient or a laboratory animal bearing renaldamage). In this method, the levels of one of the urine biomarkersdescribed above are determined before, during, and/or after thetreatment. If the urine biomarker level remains the same or decreasesover the course of the treatment, it indicates that the treatment iseffective.

Any of the urine biomarkers can also be used to monitor renal toxicityof a target agent, i.e., whether an agent induces renal damage. Thetarget agent can be any compound or composition for humanadministration. Examples include, but are not limited to, chemicalcompounds, which can be drugs (e.g., non-steroidal anti-inflammatorydrugs) or drug candidates, food products or supplements, and herbsupplements. Renal toxicity of a target agent is indicated by itsability to increase the level of a urine biomarker over time.

Also disclosed herein is a kit useful in practicing any of theabove-described methods. This kit contains at least two antibodies, onespecific to Ig-like receptor-2, e.g., capable of binding to its fragmentDFLELLVKGTVPGTEASGFDAP (SEQ ID NO:1) or any epitope contained therein,and the other specific to alpha-1 acid glycoprotein, e.g., capable ofbinding to its fragment GQEHFAHLLILRDTKTYMLAFDVNDEKNWGLS (SEQ ID NO:2)or any epitope contained therein. In one example, the kit includes twodifferent antibodies (i.e., a coating antibody and a detecting antibody)that bind to the same biomarker. Typically, the detecting antibody isconjugated with a molecule which emits a detectable signal either on itsown or via binding to another agent. The term “antibody” used hereinrefers to a whole immunoglobulin or a fragment thereof, such as Fab orF(ab′)₂ that retains antigen-binding activity. It can be naturallyoccurring or genetically engineered (e.g., single-chain antibody,chimeric antibody, or humanized antibody).

The antibodies included in the kit of this invention can be obtainedfrom commercial vendors. Alternatively, they can be prepared byconventional methods. See, for example, Harlow and Lane, (1988)Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, NewYork. To produce antibodies against a particular biomarker as listedabove, the marker, optionally coupled to a carrier protein (e.g., KLH),can be mixed with an adjuvant, and injected into a host animal.Antibodies produced in the animal can then be purified by affinitychromatography. Commonly employed host animals include rabbits, mice,guinea pigs, and rats. Various adjuvants that can be used to increasethe immunological response depend on the host species and includeFreund's adjuvant (complete and incomplete), mineral gels such asaluminum hydroxide, CpG, surface-active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, and dinitrophenol. Useful human adjuvants include BCG(bacille Calmette-Guerin) and Corynebacterium parvum. Polyclonalantibodies, i.e., heterogeneous populations of antibody molecules, arepresent in the sera of the immunized animal.

Monoclonal antibodies, i.e., homogeneous populations of antibodymolecules, can be prepared using standard hybridoma technology (see, forexample, Kohler et al. (1975) Nature 256, 495; Kohler et al. (1976) Eur.J. Immunol. 6, 511; Kohler et al. (1976) Eur J Immunol 6, 292; andHammerling et al. (1981) Monoclonal Antibodies and T Cell Hybridomas,Elsevier, N.Y.). In particular, monoclonal antibodies can be obtained byany technique that provides for the production of antibody molecules bycontinuous cell lines in culture such as described in Kohler et al.(1975) Nature 256, 495 and U.S. Pat. No. 4,376,110; the human B-cellhybridoma technique (Kosbor et al. (1983) Immunol Today 4, 72; Cole etal. (1983) Proc. Natl. Acad. Sci. USA 80, 2026, and the EBV-hybridomatechnique (Cole et al. (1983) Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc., pp. 77-96). Such antibodies can be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, IgD, and any subclassthereof. The hybridoma producing the monoclonal antibodies of theinvention may be cultivated in vitro or in vivo. The ability to producehigh titers of monoclonal antibodies in vivo makes it a particularlyuseful method of production.

Moreover, antibody fragments can be generated by known techniques. Forexample, such fragments include, but are not limited to, F(ab′)₂fragments that can be produced by pepsin digestion of an antibodymolecule, and Fab fragments that can be generated by reducing thedisulfide bridges of F(ab′)₂ fragments.

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference.

Example 1 Diagnosing Nephropathy Using Urine Leukocyte-AssociatedIg-Like Receptor-2 or Alpha-1 Acid Glycoprotein as a Biomarker

Material and Methods

(i) Subjects

The following groups of human subjects were participated in this study:

(a) healthy donors: free of diabetic mellitus with normal renalfunctions,

(b) DM patients: having type 2 diabetic mellitus, but free fromnephropathy,

(c) DN patients: having diabetic nephropathy,

(d) DN uremia patients: having DN associated with uremia,

(e) IgAN patients: having IgA nephropathy,

(f) MGN patients: having membranous glomerulonephritis,

(g) CHN patients: having nephropathy induced by Chinese herb, and

(h) CIN patients: having chronic interstitial nephritis.

Clinical characteristics of the healthy donors and the patients aresummarized in Table 1 below:

TABLE 1 Patient Characteristics Healthy DM DN DN uremia IgAN MGN CHN CINAge, mean (SD) 67.94 57.33 72.38 58.14 28.33 38.00 47.42 59.88 (12.30)(10.52) (7.44) (12.79) (12.31) (13.11) (10.43) (7.62) Female, n (%) 6 21 2 4 2 14 4 (37.5) (33.33) (12.5) (28.57) (44.44) (66.67) (73.68)(50.00) Serum Creatinine (mg/dL), 0.86 0.85 1.39 4.23 1.03 0.60 5.393.54 mean (SD) (0.14) (0.24) (0.49) (4.65) (0.48) (0.20) (5.39) (2.28)MDRD_S_GFR, mean (SD) 86.28 106.86 58.60 58.02 104.29 144.96 22.70 24.32(13.19) (70.47) (20.75) (61.53) (56.19) (55.15) (17.07) (15.91)(ii) MALDI-MS Assay

Midstream urinary samples were collected from the groups of humansubjects listed above in early morning. These urine samples from bothhealthy donors and patients, mixed with protease inhibitors, wereanalyzed by MALDI-TOF-MS. Peptide candidates that were differentiallypresented in the healthy donor group and the various patient groups wereidentified upon comparison of polypeptide patterns between each patientgroup and the healthy donor group, taking into consideration statisticalevaluation of demographic and sample parameters. These peptides werepurified, their amino acid sequences determined via routine technology.

(Iii) Western Blot Assay

Western blotting analysis was performed using antibodies specific toleukocyte-associated Ig-like receptor-2 fragment DFLELLVKGTVPGTEASGFDAP(SEQ ID NO:1) and alpha-1 acid glycoprotein fragmentGQEHFAHLLILRDTKTYMLAFDVNDEKNWGLS (SEQ ID NO:2), following routinetechnology. The results were normalized against the level of creatinineor protein in the same sample.

(iv) ELISA

Urine samples were mixed with protease inhibitors and diluted at 1:100with a dilution buffer and serum samples were diluted at 1:10. Thediluted samples were placed in ELISA plates in triplicates. Theconcentrations of leukocyte-associated Ig-like receptor-2 and alpha-1acid glycoprotein were determined via the standard sandwich ELISA methodand normalized against the level of creatinine or protein in the samesample.

Results

Via the MALDI-MS assay described above, peptides DFLELLVKGTVPGTEASGFDAP(SEQ ID NO:1) and GQEHFAHLLILRDTKTYMLAFDVNDEKNWGLS (SEQ ID NO:2) weredetected in urine samples from nephropathy patients at much higherlevels as compared to urine samples from healthy controls. SEQ ID NOs:1and 2 are fragments of leukocyte-associated Ig-like receptor-2 andalpha-1 acid glycoprotein, respectively. The positive rates of these twopeptides in the healthy donor group and in the various patient groupsare shown in Table 2 below:

TABLE 2 Positive Positive rates of rates of Patient SEQ ID NO: 2 SEQ IDNO: 1 Categories Groups Numbers N (%) N (%) Healthy Health 19  0 (0%)  1(5.3%) Diabetic DM 7  0 (0%)  2 (28.6%) Nephropathy DN 8  2 (25%)  8(100%) DN uremia 11  6 (54.5%)  8 (72.7%) Immune- IgAN 12  0 (0%)  8(66.7%) mediated MGN 3  0 (0%)  2 (66.7%) Nephropathy Interstitial CHN18 10 (55.6%) 17 (94.4%) Nephritis CIN 7  3 (42.9%)  7 (100%) Total 8521 53

The results also show that the urine levels of these two peptides innephropathy patients were not correlated with proteinuria, indicatingthat they can be used to detect kidney lesions prior to the appearanceof proteins, particularly albumin, in urine.

Further, the urine levels of these two peptides in nephropathy patientswere found to exhibit reverse correlations with GFR, indicating thatthey can serve as markers for monitoring renal function changes andnephropathy progress.

Via the ELISA and Western blot assays described above,leukocyte-associated Ig-like receptor-2 and alpha-1 acid glycoproteinwere found to be differentially presented in urine samples fromnephropathy patients (e.g., patients having Chinese herb-inducednephropathy) versus urine samples from healthy controls. See Table 3below. More specifically, presence of either protein in urine samplesfrom healthy controls was barely detectable; while a higher level of theprotein was found in urine samples from nephropathy patients. Thisresult indicates that either protein can be used as a marker fordiagnosing nephropathy.

TABLE 2 Ratios of Alpha-1 Acid Glycoprotein to Creatinine in VariousPatient Groups Healthy DM DN IgAN MGN CHN CIN DN uremia AGP/Cr (ng/mg) ×1000 2.57 2.91 29.18 15.52 139.51 19.55 51.31 97.13 mean (SD) (2.52)(1.68) (30.93) (20.87) (137.53) (39.36) (65.46) (140.60)

Example 2 Diagnosing Nephropathy Using the Combination ofLeukocyte-Associated Ig-Like Receptor-2 and Alpha-1 Acid Glycorproteinas a Biomarker

The levels of urine leukocyte-associated Ig-like receptor-2 and alpha-1acid glycorprotein from both healthy controls and nephropathy patients(including patients having diabetic nephropathy, uremia, IgAnephropathy, Chinese herb-induced nephropathy, and membranousglomerulonephritis nephropathy) were determined as described in Example1 above.

As shown in FIG. 1, the combined level of the above-mentioned twoprotein markers was much higher in all types of nephropathy patients ascompared to that in healthy controls (AUROC=0.93). This indicates thatleukocyte-associated Ig-like receptor-2 and alpha-1 acid glycorprotein,in combination, can be used as a reliable biomarker for diagnosingnephropathy with high sensitivity and specificity.

The combination of urine leukocyte-associated Ig-like receptor-2 andalpha-1 acid glycorprotein was found to be particularly reliable indetecting nephropathy induced by Chinese herb. See FIG. 2. The AUROCobtained from this study reaches 1.0, indicating that the diagnosingaccuracy is 100% when using this biomarker in diagnosing patients havingChinese herb-induced nephropathy.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

What is claimed is:
 1. A method of detecting, in a urine sample, thelevel of a fragment of leukocyte-associated Ig-like receptor-2consisting of the sequence of SEQ ID NO:1, wherein the detecting step isperformed with an immune assay.
 2. The method of claim 1, wherein theimmune assay is Western blot, ELISA, radioimmunoassay (RIA), fluorescentimmunoassay (FIA), or luminescence immunoassay (LIA).
 3. The method ofclaim 1, wherein the urine sample is obtained from a human subject. 4.The method of claim 1, wherein the urine sample is obtained from alaboratory animal.